Accumulating molecular data increasingly support an active role for adipose tissue (AT) in the development of obesity and related metabolic diseases. While a number of AT-derived signaling factors have been identified, the mechanisms by which these factors regulate AT formation remain unclear. In vivo observations in rodents have suggested that enlarged (hypertrophic) fat cells (adipocytes) induce proliferation and recruitment of new adipocytes (hyperplasia) from locally resident precursor cells. In the absence of biochemical details, this phenomenon is still controversial, especially in humans. The dual goals of this project are: (a) to investigate a hypothesized link between AT metabolism and development;and (b) to engineer an advanced AT model that will support the investigation of cell-cell signaling events in a well-defined, yet physiologically relevant experimental setting. The envisioned 3-dimensional (3D) model is a hydrogel-based construct of adipocytes, preadipocytes and vascular endothelial cells. For enhanced micro-environmental control, the 3D construct will be loaded into a micro-fluidic gradient chamber (&#956;-Gradient Chamber) supporting spatially defined chemical settings (on cellular length scales). The dual goals will be addressed through the following four specific aims. Aim 1 is to develop and characterize the 3D co-culture model. Aim 2 is to generate fluorescent reporter cells for profiling the dynamics of adipocyte- and endothelial cell-derived signaling factors. Aim 3 utilizes siRNAmediated knockdowns to characterize the effects of metabolic enzyme inhibitions on adipocyte endocrine signaling activity. The initial knockdown targets are: glucose transporter GLUT4;lipogenic enzymes ACC, FAS and AWAT;and lipolysis enzyme lipase. Selection of these targets is based on an earlier study linking adipocyte hypertrophy with metabolic flux changes (our work). A more recent study with chemical inhibitors demonstrated that down-regulating specific steps in glycolysis or fatty acid synthesis could reduce net lipid storage (our work). Aim 4 will study the inhibitors[unreadable] effects on paracrine interactions between adipocytes and neighboring endothelial cells. In vitro model and reporter system development (Aims #1 and #2) and enzyme inhibition experiments (Aims #3 and #4) will proceed along parallel tracks. While Aims #3 and #4 will ideally leverage the developments of Aims #1 and #2, the research design permits the use of currently available model systems and assay methods as backup. Throughout this project, special emphasis will be placed on comprehensively evaluating a broad range of adipocyte functions through quantitative metabolic analysis tools and advanced imaging techniques. The technical outcomes of this project should provide a broadly useful platform for controlled studies on AT intrinsic biochemical events related to the signaling functions of the tissue. The results of the planned experiments should shed new insights on the relationship between the metabolic and signaling functions of AT. Prospectively, these insights could lead to novel metabolic targets or nutritional strategies to control diseases and disorders resulting from or related to excessive AT expansion, including obesity and type 2 diabetes.